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Guide to RNA extraction
General considerations RNA is a transient molecule in any cell. Its existence is brief as the cell is constantly cleaning up RNA to prevent excessive translation. To make sure that un-needed RNAs are destroyed, the cell relies on endogenous RNase enzymes to break-down cellular RNA that is no longer required. As a result, RNase is quite pervasive, present everywhere that cells are. RNases present a serious problem for RNA extraction; they rapidly degrade RNA. RNA extraction techniques must take this into consideration and avoid exposing RNA to RNases. 'Here are some general pointers to prevent RNase contamination:' *Always wear gloves and change them frequently (whenever you touch something that might be contaminated with RNases such as skin, clothes, work surfaces, doorknobs, etc.) *Use a dedicated set of pipettors *Use RNase-free tips and tubes (Tips and tubes come in certified RNase-free varieties. These should be stored separately and should not be autoclaved but rather used straight out of the bag. Take extra care to maintain a clean stock.) *Use RNase-free reagents *Designate an RNase-free workspace and RNase-free equipment *Do not work directly over top of open tubes *Work away from vents and windows *Surfaces and equipment should be wiped down with 70% ethanol and DEPC-treated water, or with RNase-out (or a similar commercial product) *Bake glass- and metal-ware in a dry heat oven for 3-4 hours at 200 degrees Celsius. *Rinse non-disposable plasticware with 0.1M NaOH/1mM EDTA then with DEPC-treated water *Chemicals used in RNA isolation and analysis should be reserved for RNA applications and kept separate from other chemicals. Wear gloves when handling chemicals and only used baked spatulas and sterile weigh boats. *Use DEPC-treated H20 Preparing DEPC/DMPC treated water When working with RNA solutions use DEPC or DMPC treated H20. DEPC is a non-specific inhibitor of ribonucleases. DEPC also reacts with adenine so it must be inactivated by autoclaving. #'Use at 0.1% in ddH20 (1ul / ml)' #'Incubate overnight or for several hours at room temp' #'autoclave to destroy remaining DEPC' Note: Do not add DEPC to any buffers containing Trizma or mercaptans. DEPC is reactive with these products. Instead, use DEPC-treated water to make up Trizma containing buffers. Stock Solutions *'HCl, 1M, 50 ml' **Add 4.32 ml concentrated HCl to 45.69 ml H2O *'NaCl, 5M, 100 ml' **Dissolve 29.2 g NaCl in 75 ml H2O **Add 100 ul DMPC **Add H2O to 100 ml **Mix and autoclave *'NaOH, 5M, 50 ml' **Dissolve 10 g NaOH in 30 ml H2O **Add H2O to 50 ml *'LiCl, 10M, 50 ml' **Dissolve 21 g LiCl in 30 ml of water (this will take at quite a while) **Add H2O to 50 ml **Sterilize by filtration *'SDS, 10%, 50 ml' **Dissolve 5 g SDS in 30 ml water mixing slowly to avoid bubbling **Add H2O to 50 ml pouring slowly to avoid bubbling *'CTAB, 20%, 50 ml' **Dissolve 10 g CTAB in 30 ml H2O mixing slowly to avoid bubbling **Add 50 ul DMPC **Add H2O to 50 ml **Mix and autoclave *'PVP-40, 20%, 50 ml' **Dissolve 10 g PVP-40 in 30 ml H2O mixing slowly to avoid bubbling **Add H2O to 50 ml *'EDTA, 0.5M, pH 8, 50 ml' **Dissolve 9.3 g Na2EDTA-2H2O in 35 ml H2O **Adjust to pH 8 with 10M NaOH (~2.5ml) **Add 50 ul DMPC **Add H2O to 50 ml **Mix and autoclave *'Tris-HCl, 1M, pH 8, 50 ml' **Dissolve 6.05 g Tris base in 30 ml H2O **Adjust to pH 8 with concentrated HCl **Add H2O to 50 ml **Mix and autoclave *'Spermidine, 50 g/L, 10 ml' **Add 0.5 g spermidine to 8 ml H2O **Add H2O to 5 ml **Sterilize by filtration *'CTAB extraction buffer, 100 ml' **10 ml CTAB (20%) **10 ml PVP-40 (20%) **10 ml Tris-HCl (1M) **5 ml EDTA (0.5M) **40 ml NaCl (5M) **Add H2O to 97 ml **Mix and autoclave **Add 1 ml filter purified spermidine (50 g/L) **Add 3% B-mercaptoethanol immediately prior to use Solutions and reagents for extraction *'CTAB extraction buffer:' **2% CTAB (hexadecyltrimethylammonium bromide) **2% PVP (polyvinylpyrrolidinone K 30) **100 mM Tris-HCl (pH 8.0) **25 mM EDTA **2.0 M NaCl **0.5 g/L spermidine **Mix and autoclave **3% B-mercaptoethanol (added to preheated buffer prior to extraction) *'Chloroform:isoamyl alchol (24:1)' *'8 M Lithium Chloride' *'Phenol:chloroform (pH 4.7, 24:1)' *'3 M Sodium Acetate (NaOAc, pH 5.5)' *'70% Ethanol prepared with DEPC-H2O' *'100% Ethanol' Grinding tissue The most critical step in an RNA extraction is the grinding stage. It is imperative to grind the tissue thoroughly and transfer it to the extraction buffer without letting it thaw. Thawing exposes RNA to RNases which inevitably results in poor yields. Therefore, it is essential to keep the samples frozen as you grind them and to quickly and completely immerse the ground tissue in extraction buffer (which provides a protective effect). #When you take sample tissue from a specimen, quickly put it in a labelled tube and submerge that tube in liquid nitrogen to flash freeze the sample. #Precool the mortar and pestle using liquid nitrogen. When you add liquid nitrogen to the warm mortar, it will boil vigorously. When the mortar is sufficiently cold, the liquid N2 will boil less vigorously and you will probably need a thermal glove to touch it. #Once the mortar is cold, fill it about half-way with liquid nitrogen and add the frozen sample tissue. If you are having difficulty removing the sample from the tube, use a pair of forceps precooled with liquid nitrogen. #Grind the tissue using a rocking motion with the pestle being careful not to splash the ground tissue out of the mortar. Continue grinding until there the liquid N2 is almost completely evaporated. #Add more liquid N2 and repeat step 3 at least 4 times. When you are done the tissue should be a fine, homogenous powder. When you think you’re done, the sample could probably benefit from three more doses of liquid N2 and grinding. #Chill the end of a spatula by submerging it liquid nitrogen, then use it to transfer the powder into a tube containing preheated extraction buffer. #Shake/vortex immediately to fully submerge the powder, otherwise, the powder will sit on top of the extraction buffer and begin to thaw. #Do one sample at a time. Modified CTAB method for RNA extraction Unless otherwise noted, perform all reactions on ice using RNase-free tubes and tips. #For each sample preheat 1.2 ml CTAB extraction buffer (with 36 ul B-mercaptoethanol added) in a 2 ml microcentrifuge tube in a 65 degrees C water bath. #Grind 250 mg leaf tissue in liquid nitrogen and decant powder into preheated extraction buffer. #Vortex vigorously and replace in water bath. Incubate for at least 30 minutes vortexing every 5 minutes. #Centrifuge at max speed for 10 minutes and transfer the supernatant to a new 2 ml microcentrifuge tube. #Add an equal volume of chloroform:IAA (24:1), vortex for 30 second and centrifuge at max speed for 15 minutes at 4 degrees C. Transfer the aqueous phase to a new 2 ml microcentrifuge tube being careful not to disturb the interphase. #Repeat step 5 once. #Add ⅓ volume of 8M LiCl to the supernatant and mix immediately by pipetting. Incubate overnight at 4 degrees C. #Centrifuge at maximum speed for 60 minutes at 4 degrees C. #Remove supernatant and wash each pellet with 800ul of 70% ethanol prepared with DEPC-H2O. #Centrifuge at maximum speed for 10 minutes, then carefully remove the EtOH with a pipette. Repeat spin for 5 min. Remove any residual EtOH and air-dry pellets for 10 minutes. #Resuspend in 100 ul of DEPC-H2O. #Extract with equal volume phenol:chloroform, pH 4.7 (24:1). Centrifuge at max speed for 5 minutes then transfer aqueous phase to a new 1.5 ml microcentrifuge tube. #Extract with equal volumen chloroform:IAA (24:1) #Add 0.1 volumes of 3M NaOAc pH 5 and 2 volumes of 100% ethanol and precipitate for at least 30 minutes at -80 degrees C. Centrifuge for 30 min at 4 degrees C. #Remove supernatant and wash each pellet with 800ul of 70% ethanol prepared with DEPC-H2O. #Centrifuge at maximum speed for 10 minutes, then carefully remove the EtOH with a pipette. Repeat spin for 5 min. Remove any residual EtOH and air-dry pellets for 10 minutes. #Resuspend in 30 ul of DEPC-H2O. #Analyze quality and concentration RNA quality analysis The integrity and quality of an RNA sample can be determined by gel electrophoresis. This will allow you to determine whether the RNA has been degraded and will also give you some information about yield'.' #'Prepare a 1% agarose ge'l ##For one of the small gels, add 0.5 g agarose to 50 ml of 1X TAE running buffer and microwave on high for 1 minute and 30 seconds or until the agarose is fully dissolved. ##Let the solution cool until you can comfortable touch it and add 5 ul ethidium bromide. It is important to let the solution cool before adding EtBr so that you don’t breath in any EtBr vapor. ##Pour the gel and let it solidify for at least 30 minutes ##Assemble the gel in the tank, and add enough 1X TAE running buffer to cover the gel by a few millimeters. Then gently remove the comb. #'Prepare the RNA sample' ##To 2 ul of RNA sample add 8 ul DEPC treated H2O and 2 ul of 6X type III loading buffer. Mix by pipetting. ##Load 12 ul of the resulting mixture into each well. Be sure to include a size marker in one of the wells. #'Electropheresis' ##Electrophorese at 95-105 V until the bromophenol blue (the faster-migrating dye) has migrated roughly half the length of the gel. It is important not to electrophorese the gel for longer since the ethidium bromide will migrate up past the RNA. As a result, you won’t be able to see RNA that has migrated past the EtBr. #'Results' ##Visualize the gel using the gel doc. ##Intact total RNA will have sharp 18S and 28S rRNA bands. The 28S band should be roughly twice as bright as the 18S band. Partially degraded RNA will have a smeared appearance, will lack sharp ribosomal bands, or will not exhibit a 2:1 28S to 18S ratio. Completely degraded RNA will appear as a low molecular weight smear. RNA quantification Category:Lab techniques